Bulky lesions on the displaced strand stimulate DNA unwinding by dimeric UvrD-family helicases

Abstract

UvrD-family SF1A helicases play a variety of biological roles in DNA metabolism including replication, recombination, DNA repair, and conjugative plasmid transfer. The family is described by a subdomain architecture consisting of two RecA motor subdomains (1A and 2A) that each contain an auxiliary B-domain insertion (1B and 2B). Monomeric UvrD-family enzymes possess ATPase and 3-5 single-stranded DNA translocase activity but lack helicase activity. An enzyme dimer formed through a 2B-2B domain interface acts as a processive DNA helicase. A partial explanation for this observation, based on structural comparison between monomeric and dimeric DNA bound complexes, is that dimerization removes inhibitory contacts between the 2B domain and the double-stranded DNA, thus activating the helicase. However, biochemical observations reveal additional aspects of the dimeric mechanism that cannot solely be explained by movement of the 2B subdomain. Here, we present data showing that the Mycobacterium tuberculosis UvrD1 dimer interacts with the displaced strand of the duplex (i.e., the 5-3 strand in the direction of unwinding). In particular, bulky lesions on the displaced strand-including a thymine dimer-led to more efficient unwinding over a finite range of duplex lengths. These findings suggest a model for dimeric UvrD-family unwinding wherein one subunit makes intimate contacts with the displaced strand and that this interaction promotes the processive DNA unwinding unique to dimers.